CN103667188A - Method of preparing mature erythrocyte - Google Patents
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Abstract
The invention relates to a method of preparing mature erythrocyte. The method comprises a step of preparing the mature erythrocyte by using mononuclear cell to culture in vitro, wherein preferably, the mononuclear cell comes from tunica albuginea layer of cord blood or peripheral blood. The invention further relates to the mature erythrocyte prepared by using the method and a use of the mononuclear cell to preparation of the mature erythrocyte. The preparation method has the following advantages: the materials are easily obtained, and remaining part after blood is subjected to leukocyte-filtering can be used; the cost is saved, and a step of purifying CD34<+>cell is omitted.
Description
Technical field
The present invention relates to a kind of ripe erythrocytic method of preparing, specifically, the present invention relates to utilize mononuclearcell to prepare ripe erythrocytic method through vitro culture.The invention still further relates to the mature erythrocyte that utilizes described method to prepare, and mononuclearcell is for the preparation of the purposes of mature erythrocyte.
Background technology
In the century in the past, allogeneic blood transfusion (has for example become treatment severe ischemic patient, acute anemia, serious thrombocytopenia, hemophilia) help means, but, allogeneic blood transfusion causes the risk of pathophoresis (for example, hepatitis, AIDS, syphilis, malaria and other blood borne diseases) still to exist.Today, in the developed countries such as North America, West Europe and some developing country, allogeneic blood transfusion is generally all safe, and this is because the detection method of having used hypersensitivity is now come examination AIDS and other infectious diseases as nucleic acid amplification technologies.In the U.S., having a big risk of single blood transfusion infection HIV is about 1/1,000,000(McCullough, and J. is at the progress obtaining aspect cause of disease blood supply (Progress toward a pathogen-free blood supply).Clinical Infec.Dis.2003;37:88–95）。But the risk that occurs to infect by blood transfusion still exists, because infected by microbes has a window phase, within this period, detected result is negative.For example, for those emerging pathogenic agent (, various Creutzfeldt-Jakob diseases, west Nile virus, A5N1 and other avian influenza virus), also do not include at present sensing range in.In third world countries, haematogenous spreads disease generally popular, this because donor screening of blood is strict, there is blood sales phenomenon and rely on family's donor and donate blood etc. and to cause.In 2008, the whole world has gathered 93 altogether, 000, the blood of 000Ge unit, wherein approximately there is 50% blood Shi You developing country to gather, but there are 42 countries to carry out four kinds of blood transfusion infection microorganism (HIV to all blood, hepatitis B, hepatitis C and syphilis) in one or more examination, in developing country, only have 47% blood under standard Quality Control condition, detect (World Health Organization. (2010). global blood safety and operability-ultimate facts and data (Global blood safety and availability-key facts and figures), 2010).
Except the risk of pathophoresis, allogeneic donor blood is worldwide all in short state, and this shortage is by long-term existence.Because China's problem of an aging population is more and more outstanding, the crowd's proportion that is suitable for donating blood, also in continuous decline, causes blood source in short supply.In addition, donor red corpuscle can only store the time of approximately 5 weeks, is having a large amount of wounded (as natural disaster, war) to need in the situation of blood transfusion, and the problem of short supply will be more urgent.Moreover, the antigen/antibody state that allogeneic blood transfusion needs donor and acceptor to have to match, this also can cause blood supply to occur structural shortage.
Therefore, the medical science of transfusing blood at present is still faced with a lot of problems, wherein just comprises that blood imbalance between supply and demand is outstanding, blood transfusion infection still exists, clinical blood transfusion reaction happens occasionally and rare blood type patient is difficult to obtain in time suitable blood etc.This also makes the people who is engaged in blood transfusion medical research think deeply always and study how to obtain safer, more standardized blood products, wherein utilizing Stem cells cultured in vitro to produce the ripe red corpuscle that has function is exactly the target of everybody effort always, these red corpuscle can be called as the red corpuscle (cultured red blood cells, cRBC) of vitro culture.
From current present Research, what cultured and amplified in vitro red corpuscle mainly utilized is hemopoietic stem cell (hematopoietic stem cells, HSCs) and embryonic stem cell (embryonic stem cells, ESCs).
Utilizing in vitro hemopoietic stem cell to produce ripe erythrocytic method roughly comprises the steps: first to utilize cytokine as the amplification of the hemopoietic stem cells such as EPO, SCF, IL-3 and induces it to erythroid differentiation, then with EPO, stimulate it is further increased separately, the finally support by stroma cell makes cell stoning form ripe red corpuscle.Profit has successfully been turned out the ripe red corpuscle that has function in this way, and wherein more successful example is to utilize the hemopoietic stem cell (CD34 of bleeding of the umbilicus, periphery or derived from bone marrow
+cell) by adding serum-free culture and the stroma cell (mouse bone marrow or human mesenchymal stem cell) of EPO, SCF, IL-3, hydroxycorticoid, support cultivation to obtain red corpuscle, profit can make the hematopoietic stem cell expansion (2-6) * 10 of derived from cord blood in this way
6doubly, and in the cell obtaining more than 90% is all the mature erythrocyte of stoning, these cells have normal deformability and normally functioning oxyphorase (Giarratana MC, Kobari L, the people such as Lapillonne H, utilize the full ripe HRBC of the external preparation of hemopoietic stem cell (Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells) Nat.Biotechnol, 2005,23 (1): 69-74).
But with stroma cell support, cultivate and to be obviously difficult to scale operation red corpuscle in vitro, to reach the level of clinical application, therefore setting up the liquid culture system suspending the completely ripe red corpuscle that increases is according to the erythrocytic inevitable requirement of GMP standard production.Miharada etc. have set up a kind of four step liquid cultivating methods from bleeding of the umbilicus CD34
+the mature erythrocyte that increases in cell adds respectively different cytokines in these four steps, but need not any trophocyte, and result makes total amplification times of cell reach 6 * 10
5, GPA wherein
+cell (representing acidophilic normoblast and mature erythrocyte) accounts for 93.6%, the red corpuscle of stoning accounts for 50.0%(Miharada K, Hiroyama T, SudoK etc., the normoblastic effectively de-core (Efficient enucleation of erythroblasts differentiated in vitro from hematopoietic stem and progenitor cells) that hematopoietic stem/progenitor cells forms through vitro differentiation, Nat Biotechnol, 2006,24:1255-1256).Another one Testing Team utilizes SCF, EPO, IGF-1 and steroid hormone to stimulate, and also from periphery, mobilizes the CD34 of (G-CSF stimulates from peripheral blood separated)
+in cell, amplified the red corpuscle of stoning, but this method can only make tens times of cell amplifications, and wherein the red corpuscle of stoning only accounts for the 48.8%(Dorn I of total cellular score, Lazar-Karsten P, Boie S, Ribbat J etc., utilize two kinds of different cell culture systems by the people CD34 of derived from peripheral blood
+cell is bred in vitro and is divided into mature erythrocyte (In vitro proliferation and differentiation of human CD34
+cells from peripheral blood into muture red blood cells with two different cell culture systems), Transfusion, 2008,48:1122-1132.).From these results, current set up liquid culture system is from cells expanded or mature erythrocyte proportion all also cannot reach the expanding effect of supporting culture system.
Up to the present, people have utilized multiple stem cell or progenitor cell to carry out vitro culture, attempt to produce on a large scale red corpuscle, comprising the CD34 mobilizing with periphery of bleeding of the umbilicus, derived from bone marrow
+cell and human embryo stem cell, but the hematopoietic stem/progenitor of the less concern derived from peripheral blood of people because the hematopoietic stem/progenitor multiplication capacity that it is believed that derived from peripheral blood a little less than, and contained CD34 wherein
+cell proportion is lower.Yet the people such as Boehm D study discovery, the CD34 of derived from peripheral blood
+cell can increase 1.5 * 10 in the culture system without stroma cell support
6doubly, with the CD34 of derived from cord blood
+the expanding effect that cell reaches is similar.The CD34 of this explanation derived from peripheral blood
+the multiplication capacity of cell is also very strong (Boehm D, Murphy WG and Al-Rubeai M, the CD34 in human peripheral source
+cell is for the erythrocytic potential of produced in vitro (The potential of human peripheral blood derived CD34+cells for ex vivo red blood cell production), J Biotechnol.2009; 144 (2): 127-134).
Due to CD34 in peripheral blood mononuclear cell
+the content of cell is really very low, is about 0.1%, and separation of C D34 therefrom
+cell is as producing erythrocytic parent material, and not only bothersome effort, and meeting is extra increases many costs, so needs a kind of starting material source badly and enrich and the easy red corpuscle production method of preparation method.
Summary of the invention
Contriver gropes by a large amount of experiments, finally finds a kind of mononuclearcell that utilizes directly to prepare erythrocytic method as parent material, and in the time of so both can utilizing blood treatment, remaining part, as starting material, can be saved separation of C D34 again
+the step of cell.The present invention is based on above discovery and complete.
First aspect present invention relates to a kind of ripe erythrocytic method of preparing, and described method comprises utilizes mononuclearcell to prepare ripe erythrocytic step through vitro culture.
According to the method for first aspect present invention, wherein said mononuclearcell is from bleeding of the umbilicus or peripheral blood; Can utilize the direct separation of whole blood of bleeding of the umbilicus or peripheral blood to obtain mononuclearcell, from tunica albuginea layer, prepare mononuclearcell again after also can isolating tunica albuginea layer from bleeding of the umbilicus or peripheral blood.In one embodiment of the invention, described mononuclearcell is from bleeding of the umbilicus whole blood; In another embodiment of the invention, described mononuclearcell is from the tunica albuginea layer of peripheral blood.
In embodiments of the invention, wherein said mononuclearcell is that different separation obtain according to the density of cell in blood.
In embodiments of the invention, described mononuclearcell obtains mature erythrocyte through the cultivation of erythroid differentiation substratum and the de-core substratum of red corpuscle.
Second aspect present invention relates to a kind of ripe erythrocytic method of preparing, and it comprises the following steps:
A) get bleeding of the umbilicus or peripheral blood, separation obtains mononuclearcell;
B) mononuclearcell step a) being obtained is inoculated in erythroid differentiation substratum, adds hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ and erythropoietin, cultivates 8-11 days; Preferably, every 3-5 days (for example 4 days) are transferred to cell in fresh above-mentioned substratum and are continued to cultivate;
C) collect the cell in step b), be inoculated in erythroid differentiation substratum, add erythropoietin, continue to cultivate 3-5 days;
D) collect the cell in step c), with the de-core substratum of red corpuscle, continue to cultivate 7-10 days, obtain mature erythrocyte; Preferably, every 3-5 days (for example 4 days) are transferred to cell in fresh substratum and are continued to cultivate.
According to the method for second aspect present invention, wherein the mononuclearcell in step a) can utilize the direct separation of whole blood of bleeding of the umbilicus or peripheral blood to obtain, and prepares after also can isolating tunica albuginea layer from bleeding of the umbilicus or peripheral blood from tunica albuginea layer again.In one embodiment of the invention, described mononuclearcell is from bleeding of the umbilicus whole blood; In another embodiment of the invention, described mononuclearcell is from the tunica albuginea layer of peripheral blood.
In embodiments of the invention, wherein said mononuclearcell is that different separation obtain according to the density of cell in blood.According to the method for second aspect present invention, the working concentration of wherein said cytokine is that STEM CELL FACTOR 20-200ng/ml, interleukin Ⅲ are 5-20ng/mL and/or erythropoietin 2-10IU/mL.
According to the method for second aspect present invention, typical concentrations when wherein the working concentration of hydroxycorticoid is cell induction cultivation, in one embodiment of the invention, the working concentration of hydroxycorticoid is 10
-6m.
In embodiments of the invention, the concentration of described STEM CELL FACTOR is 50-200ng/ml, for example, be 50,100,200ng/ml; The concentration of described interleukin Ⅲ is 5-20ng/mL, for example, be 5,10,20ng/ml; The concentration of described erythropoietin is 3-10IU/mL, for example, be 3,5,10IU/mL.
Third aspect present invention relates to a kind of ripe erythrocytic method of preparing, and it comprises the following steps:
A) get bleeding of the umbilicus or peripheral blood, separation obtains mononuclearcell;
B) mononuclearcell step a) being obtained is inoculated in erythroid differentiation substratum, add hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and FMS sample Tyrosylprotein kinase 3 aglucons, cultivate 5～9 days (for example 7 days); Preferably, every 3-5 days (for example 4 days) are transferred to cell in fresh substratum and are continued to cultivate;
C) collect the cell in step b), be inoculated in erythroid differentiation substratum, add hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and type-1 insulin like growth factor, continue to cultivate 5～9 days (for example 7 days); Preferably, within every 2-3 days, cell is transferred in fresh substratum and continued to cultivate, make cell concn remain on 1 * 10
6below/ml;
D) collect the cell in step c), be inoculated in erythroid differentiation substratum, add erythropoietin, continue to cultivate 3～5 days;
E) collect the cell in step d), with the de-core substratum of red corpuscle, continue to cultivate 7-10 days, obtain mature erythrocyte; Preferably, every 3-5 days (for example 4 days) are transferred to cell in fresh substratum and are continued to cultivate.
According to the method for third aspect present invention, wherein the mononuclearcell in step a) can utilize the direct separation of whole blood of bleeding of the umbilicus or peripheral blood to obtain, and prepares after also can isolating tunica albuginea layer from bleeding of the umbilicus or peripheral blood from tunica albuginea layer again.In one embodiment of the invention, described mononuclearcell is from bleeding of the umbilicus whole blood; In another embodiment of the invention, described mononuclearcell is from the tunica albuginea layer of peripheral blood.
In embodiments of the invention, wherein said mononuclearcell is that different separation obtain according to the density of cell in blood.
According to the method for third aspect present invention, the working concentration of wherein said cytokine is that STEM CELL FACTOR 20-200ng/ml, interleukin Ⅲ are 5-20ng/mL and/or erythropoietin 2-10IU/mL.In embodiments of the invention, the concentration of described STEM CELL FACTOR is 50-200ng/ml, for example, be 50,100,200ng/ml; The concentration of described interleukin Ⅲ is 5-20ng/mL, for example, be 5,10,20ng/ml; The concentration of described erythropoietin is 3-10IU/mL, for example, be 3,5,10IU/mL.
According to the method for third aspect present invention, wherein described in step b), the working concentration of cytokine is: 2-20ng/mL bone morphogenetic protein 4,5-20ng/mL interleukin-11 and/or 20-100ng/mL Flt3 aglucon.In embodiments of the invention, the concentration of described bone morphogenetic protein 4 is 5-20ng/ml, for example, be 5,10,20ng/ml; The concentration of described interleukin-11 is 5-20ng/mL, for example, be 5,10,20ng/ml; The concentration of described Flt3 aglucon is 20-100ng/ml, for example, be 20,40,100ng/ml.
According to the method for third aspect present invention, wherein described in step c), the working concentration of cytokine is: 5-20ng/mL bone morphogenetic protein 4,5-20ng/mL interleukin-11 and/or 20-200ng/mL type-1 insulin like growth factor.In embodiments of the invention, the concentration of described bone morphogenetic protein 4 is 5-20ng/ml, for example, be 5,10,20ng/ml; The concentration of described interleukin-11 is 5-20ng/mL, for example, be 5,10,20ng/ml; The concentration of described type-1 insulin like growth factor is 20-200ng/mL, for example, be 20,40,100,200ng/ml.
According to the method for third aspect present invention, typical concentrations when wherein the working concentration of hydroxycorticoid is cell induction cultivation, in one embodiment of the invention, the working concentration of hydroxycorticoid is 10
-6m.
Fourth aspect present invention relates to according to the present invention the first mature erythrocyte preparing to method described in third aspect any one.
In embodiments of the invention, the mean corpuscular volume of described mature erythrocyte (MCV) is 108～123fl; In embodiments of the invention, the average content of hemoglobin (MCH) of described mature erythrocyte is 31～37pg; In embodiments of the invention, the maximum distortion index (DI of described mature erythrocyte
max) be 0.42～0.48; In embodiments of the invention, the content of the G 6 PD of described mature erythrocyte is 31～36U/g Hb; In embodiments of the invention, the pyruvate kinase content of described mature erythrocyte is 69～80U/gHb.
Fifth aspect present invention relates to mononuclearcell in the blood purposes in preparing mature erythrocyte.
According to the purposes of fifth aspect present invention, mononuclearcell wherein can utilize the direct separation of the whole blood of bleeding of the umbilicus or peripheral blood to obtain, and prepares after also can isolating tunica albuginea layer from bleeding of the umbilicus or peripheral blood from tunica albuginea layer again.In one embodiment of the invention, described mononuclearcell is from bleeding of the umbilicus whole blood; In another embodiment of the invention, described mononuclearcell is from the tunica albuginea layer of peripheral blood.
In embodiments of the invention, described mononuclearcell is that different separation are mentioned according to the density of cell in blood.
Sixth aspect present invention relates to cell factor composition, and it comprises hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ and erythropoietin; Preferably, it also comprises bone morphogenetic protein 4, interleukin-11 and FMS sample Tyrosylprotein kinase 3 aglucons, or preferably, it also comprises bone morphogenetic protein 4, interleukin-11 and type-1 insulin like growth factor.
In one embodiment of the invention, described cell factor composition is comprised of hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ and erythropoietin; In another embodiment of the invention, described cell factor composition is comprised of hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and FMS sample Tyrosylprotein kinase 3 aglucons; In another embodiment of the invention, described cell factor composition is comprised of hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and type-1 insulin like growth factor.
The cell factor composition that the invention still further relates to sixth aspect present invention any one is being prepared the purposes in mature erythrocyte by mononuclearcell.
Below the present invention is described further.
Term " mononuclearcell " in this article refers to the cell in blood with single core, comprises lymphocyte and monocyte, also comprises hemopoietic stem cell and progenitor cell.Can prepare mononuclearcell by the conventional method in this area, for example, can be by erythrocyte cracked liquid (being mixed by the bicarbonate of ammonia of 790mg/L and the ammonium chloride of 7.7g/L) splitting erythrocyte with separated mononuclearcell.In the present invention, mononuclearcell is that different separation obtain according to the density of cell in blood.It can utilize the direct separation of whole blood of bleeding of the umbilicus or peripheral blood to obtain, and prepares after also can isolating tunica albuginea layer from bleeding of the umbilicus or peripheral blood from tunica albuginea layer again.In one embodiment of the invention, described mononuclearcell is from bleeding of the umbilicus whole blood; In another embodiment of the invention, described mononuclearcell is from the tunica albuginea layer of peripheral blood.By separated tunica albuginea layer, separablely go out Red Blood Cells Concentrate and blood plasma for component blood transfusion.
In embodiments of the invention, separation method is Ficoll-hypaque(dextran-urografic acid methylglucamine salt) density gradient centrifugation, because the proportion of each formed elements there are differences in blood, be therefore able to separation.Red corpuscle and granulocyte density are greater than layering liquid, are deposited on the pipe end because red corpuscle runs into Ficoll aggegation bunchiness money shape simultaneously.Thrombocyte is suspended in blood plasma because density is little, have the mononuclearcell suitable with the liquid-tight degree of layering only intensive in the interface of plasma layer and layering liquid, be tunica albuginea shape (buffy coat), draw this confluent monolayer cells and pass through washing the centrifugal resuspended mononuclearcell that obtains.In embodiments of the invention, get peripheral blood tunica albuginea confluent monolayer cells or bleeding of the umbilicus whole blood dilutes with phosphate buffered saline buffer, then use lymphocyte separation medium separated, draw mononuclearcell, with phosphate buffer wash cell, obtain mononuclearcell.
Term " hemopoietic stem cell " refers to that hemopoietic stem cell refers to the stem cell in marrow, there is self-renewal capacity and can be divided into various hemocyte precursor cells, the various blood cell compositions of final generation, comprise red corpuscle, white corpuscle and thrombocyte, and they also can be divided into various other cells.Hemopoietic stem cell has two key characters: one, self or the of self-replication capacity of height; Its two, can be divided into all types of hemocytes.
Term " progenitor cell " refers under the adjusting of certain microenvironment and some factor, proliferation and differentiation is the progenitor cell of all kinds of hemocytes, also referred to as hemopoietic progenitor cell (hematopoietic progenitor cells, HPCs), it is also a kind of quite original cell with multiplication capacity, but lost Multidirectional Differentiation ability, can only be to one or several blood cell line directional proliferation differentiation, therefore also claim committed stem cell (committed stem cell).For example comprise erythron hemopoietic progenitor cell, neutrophil leucocyte-macrophage system hemopoietic progenitor cell or megakaryocytic series hemopoietic progenitor cell.
Term " tunica albuginea layer " in this article refers to the component that anticoagulation forms after natural subsidence, centrifugal or density gradient centrifugation, mainly white corpuscle and thrombocyte, consists of.Anticoagulation after centrifugal, can form the blood plasma on upper strata, the red corpuscle of lower floor and very thin one deck white membranoid substance between the two account for 1% of blood cumulative volume, is called as tunica albuginea layer.
Term " bleeding of the umbilicus " is stayed the blood in placenta and umbilical cord after in this article referring to fetal birth, is wherein rich in hemopoietic stem cell and progenitor cell.
Term " mature erythrocyte " in this article refers to the maximum a kind of hemocyte of quantity in blood, is also in vertebrates body, by blood, to transport the topmost medium of oxygen simultaneously, also has immunologic function simultaneously.Ripe red corpuscle is seedless, this means that they have lost DNA.Red corpuscle does not have plastosome yet, and they synthesize energy by glucose.
Term " STEM CELL FACTOR " in this article refers to the part of proto-oncogene c-kit expression product, mainly by marrow stromal cell, is produced, and has two kinds of forms of secretor type and transmembrane in body.Activated STEM CELL FACTOR is homodimer, and can stimulate differentiation of stem cells is different pedigree hemocytes, and stimulates proliferation of mast cells.
Term " interleukin Ⅲ " in this article refers to by the CD4 activating
+a kind of cytokine that T cell produces, its Main Function, for promoting directed differentiation and the propagation of pluripotential hemopoietic stem cell in marrow, produces various types of hemocytes.
Term " erythropoietin " in this article refers to a kind of cytokine of red corpuscle precursor cell secretion in marrow.In human body environment, it is produced by liver and kidney, is the most important cytokine that regulates erythropoiesis quantity, and it can stop CFU-E apoptosis and induce it to carry out mitotic division, therefore can accelerate the propagation of CFU-E, greatly improve erythrocytic generation quantity.
Term " lymphocyte separation medium " can be lymphocyte separation medium well known in the art, for example, can be HISTOPAQUE-1077(Sigma company).In the art, the composition of lymphocyte separation medium is consistent, as referred in embodiments of the invention the mixture being formed by dextran and urografic acid methylglucamine salt, a kind of according to cell density difference, universal gravity constant by centrifugal generation, carry out the common agents of the separation and purification of cell, its density is 1.13.
Term " erythroid differentiation substratum " refers to and is conducive to the propagation of hemopoietic stem cell and progenitor cell and to the substratum of erythroid differentiation, it can be according to the conventional formulation of this area and method preparation.In this article refer to by serum free medium, foetal calf serum and various small molecules, formed for cultivating erythrocytic a kind of substratum.The serum free medium that wherein serum free medium is cultivated for being applicable to hematopoietic cell can be for example StemSpan SFEM(Stemcell Technologies Inc.), LONZA X-VIVO 15(Lonza) or
sFM(Life Technologies).Small molecules wherein for example can comprise tocopherol, linolic acid, cholesterol, human transferrin, recombinant human insulin, thanomin, 3-mercaptoethanol, heparin sodium, ferrous citrate, vitamins C, glutamine that ion is saturated, in embodiments of the invention, described small molecules comprises tocopherol, linolic acid, cholesterol, human transferrin, recombinant human insulin, thanomin, 3-mercaptoethanol that ion is saturated.
Term " red corpuscle takes off core substratum " refers to that being conducive to late period normoblast breaks up and complete the substratum of de-nuclear process to mature erythrocyte, and it can be according to the conventional formulation of this area and method preparation.In this article refer to by IMDM, foetal calf serum and various small molecules, formed for cultivating erythrocytic a kind of substratum.IMDM wherein can substitute with other conventional substratum of this area, for example alpha-MEM or DMEM, small molecules wherein for example can comprise adenosine, D-MANNOSE, Sodium phosphate dibasic, mifepristone, thioglycerin, Pluronic F68(Sigma company), heparin sodium, in embodiments of the invention, described small molecules comprises adenosine, D-MANNOSE, Sodium phosphate dibasic and mifepristone.
In the present invention, " concentration " or " working concentration " typically refers to the final concentration of this cytokine.
In the present invention, the density that each phase cell is cultivated should maintain 1 * 10 conventionally
6below/ml, to guarantee the normal growth of cell.
In the present invention, the mature erythrocyte preparing can carry out form, structure and function is identified, for example comprise cell centrifugation smear staining, fluorescent antibody staining flow cytometry analysis, content of hemoglobin mensuration, G 6 PD and pyruvate kinase assay etc.
The beneficial effect of the invention
The present invention directly utilizes the mononuclearcell in blood to prepare mature erythrocyte, and the method has the following advantages: 1) can utilize remainder after blood treatment as the source of mononuclearcell, take full advantage of waste; 2) saved purifying CD34
+the step of cell, not only can be cost-saving, and can avoid the CD34 that causes because of purifying
+cell loss; 3) erythrocytic yield is higher, according to initial mononuclearcell number, calculates, and the mature erythrocyte number obtaining increases approximately 1000 times of left and right, due to CD34 in peripheral blood mononuclear cell
+the content of cell very low (approximately 0.1%), being therefore equivalent to amplification times has reached 10
6.
Accompanying drawing explanation
Fig. 1 is that peripheral blood mononuclear cell is produced erythrocytic cell growth curve;
Fig. 2 is the viable cell form (400 *) in peripheral blood mononuclear cell amplification in vitro process;
The morphological feature of cell and After Enucleation when Fig. 3 is different cultivated days (1000 * Wrights-Giemsa dyeing);
Fig. 4 is the different developmental phases (Pro-E: erythroblast of erythroid cells; Baso-E: basophilic erythroblast; Poly-E: polychromatophilia normoblast; Ortho-E: just dying normoblast; Enucleated: stoning red corpuscle);
Fig. 5 is the shared ratio of erythroid cells in each etap in the culture of different number of days;
Fig. 6 is differentiation and the After Enucleation of facs analysis cell; In the cell that wherein A is different cultivated days, the ratio of erythroid cells changes (anti-glycophorin A antibody positive); B is that the ratio of cell stoning stage enucleate cell changes (anti-glycophorin A antibody positive and Syto dye 16 feminine genders);
Fig. 7 is the variation of colony forming cell proportion in cell cultivation process;
Fig. 8 is the CFU-E growth curve in PBMC proliferation atomization.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example is only for the present invention is described, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: the preparation of erythroid differentiation substratum
Preparation 100mL erythroid differentiation substratum needs following composition:
Embodiment 2: the preparation of the de-core substratum of red corpuscle
The de-core substratum of preparation 100mL red corpuscle needs following composition:
| Composition | Storage liquid concentration | Final concentration | Volume (ml) |
| IMDM（Invitrogen） | 93.4 | ||
| Foetal calf serum | 0.5% | 0.5 | |
| Adenosine | 0.14g/ml | 0.14mg/ml | 0.1 |
| D-MANNOSE | 1.457g/ml | 14.57mg/ml | 1 |
| Sodium phosphate dibasic | 0.94g/ml | 0.94mg/ml | 0.1 |
| Mifepristone | 1mM | 1μM | 0.1 |
Embodiment 3: the separation of peripheral blood mononuclear cell
Peripheral blood EDTA anti-freezing, gets anticoagulation, and 800g can be divided into three layers after centrifugal 20 minutes, is respectively upper serum, lower floor's red corpuscle and middle tunica albuginea layer, and taking-up tunica albuginea layer is wherein for separating of mononuclearcell.Tunica albuginea after peripheral blood filter is white 1 * PBS(pH=7.4 for layer, lower same) press 1:1 dilution, be then layered on carefully lymphocyte separation medium Histopaque
tM(Sigma, cat#10771) upper (volume ratio is 1:1), centrifugal 30 minutes of 2500rpm, is inserted into cloud and mist layer with capillary vessel, draws mononuclearcell.Transfer in another test tube, add 1 * PBS of 5 times of volumes, 1500rpm * 10 minute, washed cell twice, re-suspended cell, is mononuclearcell suspension.
Embodiment: 4: the separation of Cord Blood Mononuclear Cell
Anti-freezing bleeding of the umbilicus is for separating of mononuclearcell.1 * PBS(pH=7.4 for bleeding of the umbilicus, lower same) press 1:1 dilution, be then layered on carefully lymphocyte separation medium Histopaque
tM(Sigma, cat# 10771) upper (volume ratio is 1:1), centrifugal 30 minutes of 2500rpm, is inserted into cloud and mist layer with capillary vessel, draws mononuclearcell.Transfer in another test tube, add 1 * PBS of 5 times of volumes, 1500rpm * 10 minute, washed cell twice, re-suspended cell, is mononuclearcell suspension.
Embodiment 5: utilize peripheral blood mononuclear cell to produce red corpuscle
1 * 10
6it is 10 that/ml mononuclearcell (utilizing embodiment 3 method preparations) is used erythroid differentiation culture medium culturing, the cytokine of interpolation
-6m hydroxycorticoid (Ca#:H0135, Sigma), 100ng/ml STEM CELL FACTOR (Ca#PHC2116, Biosource), 10ng/ml interleukin Ⅲ (Ca#PHC0031, Biosource), and 5IU/ml erythropoietin (Ca#PHC2054, Invitrgen), 5%CO
2, 37 ℃ cultivate 8 days; In the time of the 4th day, a cell culture is diluted to 4 parts and cultivates containing continuing in the fresh red blood cell division culture medium substratum of above-mentioned cytokine; In the time of the 8th day, with erythroid differentiation culture medium culturing base, cell dilution is become to 5 * 10
4the concentration of/ml, the cytokine of interpolation is 5IU/ml erythropoietin, 5%CO
2, 37 ℃ cultivate 3 days; In the time of the 11st day, with de-core substratum, cell dilution is become to 2 * 10
5the concentration of/ml, does not add any cytokine, 5%CO
2, 37 ℃ cultivate 10 days, within every 4 days, change liquid once, cultivate harvested cell while finishing, phosphate buffer 1 * PBS(pH=7.4) washed twice, cell is identified for form, structure and function.
Embodiment 6: utilize peripheral blood mononuclear cell to produce red corpuscle
By 2 * 10
5/ ml peripheral blood mononuclear cell (PB MNCs) is inoculated in erythroid differentiation substratum, and the cytokine of interpolation comprises 10
-6m hydroxycorticoid, 100ng/ml STEM CELL FACTOR, 40ng/ml FMS sample Tyrosylprotein kinase 3 aglucons (fms-like tyrosine kinase 3ligand, Flt3-L, Ca#PHC9411, Biosource), 10ng/ml bone morphogenetic protein 4(Bone Morphogenetic protein-4, BMP4, C a#PHC9534, Biosource), 10ng/mLIL-3,10ng/mlIL-11(Ca#PHC0115, Biosource) and 5U/ml erythropoietin (Erythropoietin, EPO).Within the 4th day, be to change fresh red blood cell division culture medium, the cytokine of interpolation is identical.Second step (8-14 days), cell is comprising 10
-6m hydroxycorticoid, 100ng/3ml SCF, 40ng/ml type-1 insulin like growth factor (Insulin-like growth factor 1, IGF-1, Ca#PHG0071,
in the erythroid differentiation substratum of 10ng/ml BMP4,10ng/mL IL-3,10ng/ml IL-11 and 5U/ml EPO, cultivate, every 2-3 days changes a subculture, makes cell concn remain on 10
6below/ml.The 3rd step (15-17 days), changes fresh culture, and substratum is the erythroid differentiation substratum that comprises 5IU/ml EPO.The 4th step (18-24 days), cell continues to cultivate, and substratum is not for adding the de-core substratum of any cytokine.Cultivate harvested cell while finishing, phosphate buffer 1 * PBS(pH=7.4) washed twice, cell is identified for form, structure and function.Fig. 1 is that peripheral blood mononuclear cell is produced erythrocytic cell growth curve, and Fig. 2 is the viable cell form (400 *) in peripheral blood mononuclear cell amplification in vitro process.Can find out, peripheral blood mononuclear cell is along with the increase of cultivated days, cell fast breeding, and when the 4th EOS, cell absolute number increases by 966 ± 1002 times, from 9 * 10
5individual cell amplification goes out 6.9 ± 7.1 * 10
8individual cell.And the mononuclearcell that starts inoculation is similar to lymphocyte, after cultivating, mature cell wherein (as lymphocyte, monocyte, granulocyte etc.) can be dead gradually, and progenitor cell can survive and amplification gradually, in the time of the 3rd day, most cells is all dead, and during by the 5th day, visible large cell occurs, the progenitor cell proliferation that these cells are survived forms, during by the 8th day, in culture, be all this maxicell substantially, and cell count raise fast.These results all illustrate utilizes peripheral blood mononuclear cell can amplify a large amount of red corpuscle.
The impact of table 1 different cytokines combination on red corpuscle amplification times
Total cellular score when wherein amplification times=cultivation finishes/the start cell count of inoculation
Embodiment 8: cellular form Epidemiological Analysis:
For the morphological change in monitoring peripheral blood mononuclear cell proliferation and differentiation process, get according to embodiment 6 methods and cultivate each stage (the 7th, 14,17,20 and 24 days) the cell centrifugation smear obtaining, then use Hema 3system(Fisher Scientific Company L.L.C., Cat#122-911) dyeing, by the specification sheets operation of producer, micro-Microscopic observation is also taken pictures, and result as shown in Figure 3-Figure 5.The morphological feature of cell and After Enucleation when Fig. 3 is different cultivated days (1000 *; Wrights-Giemsa dyeing).Fig. 4 is the different developmental phases (Pro-E: erythroblast of erythroid cells; Baso-E: basophilic erythroblast; Poly-E: polychromatophilia normoblast; Ortho-E: just dying normoblast; Enucleated: stoning red corpuscle).Fig. 5 is the shared ratio of erythroid cells in each etap in the culture of different number of days.
Can find out, increase along with cultivated days, cell from erythroblast (Proerythroblast) to basophilic erythroblast (Basophilic erythroblast), polychromatophilia normoblast (Polychromatic erythroblast), just dye normoblast (Orthochromatic erythroblast) differentiation, last most cells is divided into the red corpuscle of stoning, and its form is similar to normoerythrocyte.According to cell picture, various cells are carried out to differential count, when result is presented at the 7th day, approximately having 13% cell is erythroblast, and during by the 14th day, approximately 90% cell is basophilic erythroblast, and along with the further differentiation of cell, during by the 20th day, most cells becomes and just dyes normoblast and stoning red corpuscle (approximately 40%), and when cultivate finishing, the erythrocytic ratio of stoning reaches approximately 89%.These presentation of results are cultivated by four steps, and the progenitor cell in peripheral blood mononuclear cell can become mature erythrocyte by proliferation and differentiation, and reach higher stoning ratio.
Embodiment 9:FACS analyzes:
Results are washed once with 1 * PBS/2%FBS after cultivating according to embodiment 6 methods each phase cell obtaining, are then resuspended in 100 μ l 1 * PBS/2%FBS, interpolation 1 μ l 1:100 dilution
(Invitrogen, Cat#S7578, be used for the karyocyte that dyes) and glycophorin A antibody (anti-glycophorin A-PE, eBioscience, Cat#12-9987-82, for the erythroid cells that dyes), hatch 20 minutes for 4 ℃, then use 1 * PBS/2%FBS washed twice, be resuspended in 500 μ l PBS/2%FBS facs analysis.
Glycophorin A (glycophorin A) is the mark of more ripe erythroid cells, first appears at the basophilic erythroblast stage, and its expression level raises gradually along with the maturation of cell.Can utilize the cell of the different cultivated days of antibody staining of anti-glycophorin A, then by FACS, analyze mononuclearcell to the differentiation situation of erythroid cells, result as shown in Figure 6.Fig. 6 is differentiation and the After Enucleation of facs analysis cell, and in the cell that wherein Fig. 6 A is different cultivated days, the ratio of erythroid cells changes (anti-glycophorin A antibody positive).Can find out, along with the increase of cultivated days, glycophorin A positive cell percentage improves constantly, and during by 17 days, almost 100% cell is all the glycophorin A positive, and the most cells in this explanation culture have been divided into erythroid cells.
Utilize
(this dyestuff can be combined with DNA, and green-emitting fluorescence, for the karyocyte that dyes) and glycophorin A antibody staining, analyze the shared ratio of enucleate cell by FACS, in the erythroid cells of the glycophorin A positive
what dyeing was negative is stoning red corpuscle.From Fig. 6 B, can find out, in the time of the 17th day, it is seedless only having a small amount of cell, but along with the further maturation of cell, akaryotic ratio raises rapidly, during by the 21st day, reach approximately 60%, and when cultivate finishing the 24th day, akaryotic ratio can reach 90% left and right.
Embodiment 10: clone forms and analyzes
In order to analyze the content of mononuclearcell progenitor cell in proliferation and differentiation process, get the cell of the 0th, 4,7,10 and 14 days, get respectively according to embodiment 6 methods cultivate 0,4,7,10 and 14 day cell analysis its to form CFU-E(red be colony forming cell), the quick-fried formula colony forming cell of BFU-E() and CFU-GM(CFU-GM) ability, substratum is
(Ca#04034, Stem Cell Technologies), the specification sheets providing according to Stem Cell Technologies operates.Fig. 7 is the variation of colony forming cell proportion in cell cultivation process; Fig. 8 is the CFU-E growth curve in PBMC proliferation atomization.
Found that in whole culturing process, cell can only form CFU-E and BFU-E, and do not have CFU-GM colony to form, this illustrate our culture system just impel cell to red be direction differentiation.Along with the increase of cultivated days, the number of CFU-E increases gradually, and the number of BFU-E reached peak value in the time of the 7th day, then declines gradually, illustrates that these BFU-E progenitor cells are divided into CFU-E cell gradually.
Embodiment 11: red blood cell structure and Function Identification
Get and cultivate the red corpuscle obtaining while finishing, utilize red blood cell count(RBC) instrument to measure mean corpuscular volume (MCV), average content of hemoglobin (MCH).Red blood cell suspension, in 4% polyvinylpyrrolidonesolution solution, then increases the osmotic pressure (from 60 to 450mosm) of solution gradually, records its laser diffraction pattern, according to its detected value, can obtain deformation index (DI).Reference report (Giarratana MC; Kobari L; Lapillonne H, et al.Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells.Nat.Biotechnol.2005; 23 (1): method 69-74.), by the level of spectrophotometry Red Blood Cells Glucose 6 phosphate dehydrogenases and pyruvate kinase.Red blood cell structure and the Function Identification of different cytokines combinations produce the results are shown in Table 2.
The red blood cell structure of table 2 different cytokines combinations produce and Function Identification result
While utilizing combination of cytokines 5 to cultivate red corpuscle, cultivating the erythrocytic mean corpuscular volume (MCV) obtaining after 24 days is that 118 ± 4fl(normoerythrocyte is 80-100fl), illustrate that the erythrocyte volume obtaining is slightly large.Average content of hemoglobin (MCH) is that 36 ± 1.2pg(normal value is 27-31pg), slightly higher than normal hematocrit level.Erythrocytic maximum distortion index (DI
max) be 0.46, approach normoerythrocyte level.The content of G 6 PD is 35 ± 3U/g Hb, the content of pyruvate kinase is 75 ± 6U/g Hb, illustrate that these cells are consistent with the characteristic of Young erythrocyte, can reduced glutathione, maintain the level of ATP, avoid 2,3-DPG to gather, and the aglucon avidity of gathering meeting reduction oxyphorase of 2,3-DPG.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (12)
1. prepare a ripe erythrocytic method, described method comprises utilizes mononuclearcell to prepare ripe erythrocytic step through vitro culture.
2. the process of claim 1 wherein that described mononuclearcell is from bleeding of the umbilicus or peripheral blood; Preferably, described mononuclearcell is from the tunica albuginea layer of bleeding of the umbilicus or peripheral blood.
3. prepare a ripe erythrocytic method, it comprises the following steps:
A) get bleeding of the umbilicus or peripheral blood, separation obtains mononuclearcell;
B) mononuclearcell step a) being obtained is inoculated in erythroid differentiation substratum, adds hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ and erythropoietin, cultivates 8-11 days;
C) collect the cell in step b), be inoculated in erythroid differentiation substratum, add erythropoietin, continue to cultivate 3-5 days;
D) collect the cell in step c), with the de-core substratum of red corpuscle, continue to cultivate 7-10 days, obtain mature erythrocyte.
4. prepare a ripe erythrocytic method, it comprises the following steps:
A) get bleeding of the umbilicus or peripheral blood, separation obtains mononuclearcell;
B) mononuclearcell step a) being obtained is inoculated in erythroid differentiation substratum, add hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and FMS sample Tyrosylprotein kinase 3 aglucons, cultivate 5～9 days;
C) collect the cell in step b), be inoculated in erythroid differentiation substratum, add hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ, erythropoietin, bone morphogenetic protein 4, interleukin-11 and type-1 insulin like growth factor, continue to cultivate 5～9 days;
D) collect the cell in step c), be inoculated in erythroid differentiation substratum, add erythropoietin, continue to cultivate 3～5 days;
E) collect the cell in step d), with the de-core substratum of red corpuscle, continue to cultivate 7-10 days, obtain mature erythrocyte.
5. claim 3 or 4 method, be wherein to obtain mononuclearcell from the tunica albuginea layer separation of bleeding of the umbilicus or peripheral blood in step a).
6. claim 3 or 4 method, the working concentration of wherein said STEM CELL FACTOR is that the working concentration of 20-200ng/ml, interleukin Ⅲ is that the working concentration of 5-20ng/mL and/or erythropoietin is 2-10IU/mL.
7. the method for claim 4, wherein described in step b), the working concentration of bone morphogenetic protein 4 is that the working concentration of 2-20ng/mL, interleukin-11 is that the working concentration of 5-20ng/mL and/or Flt3 aglucon is 20-100ng/mL.
8. the method for claim 4, wherein described in step c), the working concentration of bone morphogenetic protein 4 is that the working concentration of 5-20ng/mL, interleukin-11 is that the working concentration of 5-20ng/mL and/or type-1 insulin like growth factor is 20-200ng/mL.
9. the mature erythrocyte preparing according to method described in claim 1-8 any one.
10. the purposes of the mononuclearcell in blood in preparing mature erythrocyte.
11. cell factor compositions, it comprises hydroxycorticoid, STEM CELL FACTOR, interleukin Ⅲ and erythropoietin; Preferably, it also comprises bone morphogenetic protein 4, interleukin-11 and FMS sample Tyrosylprotein kinase 3 aglucons, or preferably, it also comprises bone morphogenetic protein 4, interleukin-11 and type-1 insulin like growth factor.
The cell factor composition of 12. claims 11 is being prepared the purposes in mature erythrocyte by mononuclearcell.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877965A (en) * | 2015-05-27 | 2015-09-02 | 赛欧帕克（江苏）干细胞生物工程有限公司 | Method for preparing mature erythrocyte |
| CN109652369A (en) * | 2019-02-28 | 2019-04-19 | 西湖生物医药科技(杭州)有限公司 | Utilize the method and preparation of peripheral blood external preparation mature erythrocyte |
| CN109722414A (en) * | 2017-10-27 | 2019-05-07 | 博雅辑因（北京）生物科技有限公司 | It is a kind of efficiently to prepare the method for mature erythrocyte and be used to prepare the culture medium of mature erythrocyte |
| CN110982779A (en) * | 2019-12-25 | 2020-04-10 | 广州市天河诺亚生物工程有限公司 | Method for improving utilization rate of umbilical cord blood |
| CN114317431A (en) * | 2022-01-03 | 2022-04-12 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting differentiation of human hematopoietic stem/progenitor cells to erythroid line |
| WO2022089606A1 (en) * | 2020-10-30 | 2022-05-05 | Westlake Therapeutics (Hangzhou) Co. Limited | Methods for preparing mature red blood cells in vitro |
-
2012
- 2012-09-21 CN CN201210355560.XA patent/CN103667188B/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| CAIHONG QIU等: "Globin switches in yolk sac–like primitive and fetal-like definitive red blood cells produced from human embryonic stem cells", 《BLOOD》 * |
| VLADISLAV M. SANDLER等: "Reprogramming of Embryonic Human Fibroblasts into Fetal Hematopoietic Progenitors by Fusion with Human Fetal Liver CD34+ Cells", 《PLOS ONE》 * |
| 习佳飞 等: "诱导扩增脐带血单个核细胞来源的红系祖细胞及其保存", 《中国实验血液学杂志》 * |
| 何秋山 等: "脐血单个核细胞体外红系定向扩增和分化的研究", 《临床血液学杂志》 * |
| 贾延军 等: "利用外周血单个核细胞生产成熟红细胞", 《中国实验血液学杂志》 * |
| 贾延军 等: "利用外周血单个核细胞生产成熟红细胞", 《中国输血杂志》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104877965A (en) * | 2015-05-27 | 2015-09-02 | 赛欧帕克（江苏）干细胞生物工程有限公司 | Method for preparing mature erythrocyte |
| CN109722414A (en) * | 2017-10-27 | 2019-05-07 | 博雅辑因（北京）生物科技有限公司 | It is a kind of efficiently to prepare the method for mature erythrocyte and be used to prepare the culture medium of mature erythrocyte |
| CN109652369A (en) * | 2019-02-28 | 2019-04-19 | 西湖生物医药科技(杭州)有限公司 | Utilize the method and preparation of peripheral blood external preparation mature erythrocyte |
| CN111154719A (en) * | 2019-02-28 | 2020-05-15 | 西湖生物医药科技(杭州)有限公司 | Method for preparing mature red blood cells in vitro by using peripheral blood and preparation |
| WO2020172915A1 (en) * | 2019-02-28 | 2020-09-03 | 西湖生物医药科技(杭州)有限公司 | Method for using peripheral blood for in-vitro preparation of mature red blood cells, and preparation |
| WO2020173466A1 (en) * | 2019-02-28 | 2020-09-03 | Westlake Therapeutics (Hangzhou) Co. Limited | Method for preparing mature red blood cells in vitro using peripheral blood |
| CN111154719B (en) * | 2019-02-28 | 2024-03-01 | 西湖生物医药科技(杭州)有限公司 | Method for preparing mature red blood cells in vitro by using peripheral blood and preparation |
| CN110982779A (en) * | 2019-12-25 | 2020-04-10 | 广州市天河诺亚生物工程有限公司 | Method for improving utilization rate of umbilical cord blood |
| CN110982779B (en) * | 2019-12-25 | 2020-12-29 | 广州市天河诺亚生物工程有限公司 | Method for improving utilization rate of umbilical cord blood |
| WO2022089606A1 (en) * | 2020-10-30 | 2022-05-05 | Westlake Therapeutics (Hangzhou) Co. Limited | Methods for preparing mature red blood cells in vitro |
| CN114317431A (en) * | 2022-01-03 | 2022-04-12 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting differentiation of human hematopoietic stem/progenitor cells to erythroid line |
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